2,4-Diamino-5-benzylpyrimidines

ABSTRACT

This invention provides 2,4-diamino-5-benzylpyrimidines represented by the formula ##STR1## wherein R 1  is H or loweralkyl; R 2  is H, loweralkyl or R 3  CO-- wherein R 3  is H, alkyl, or cycloalkyl-lowerakyl; and X is a halogen; and the acid addition salts thereof. 
     These pyrimidine compounds are useful as antibacterial agents, and as potentiators of the anti-bacterial activity of sulfonamides.

This is a division of application Ser. No. 601,118, filed July 31, 1975now U.S. Pat. No. 4,008,236 issued Feb. 15, 1977.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to 2,4-diamino-5-benzylpyrimidines characterizedby formula (I): ##STR2## wherein R₁ is H or loweralkyl; R₂ is H,loweralkyl or R₃ CO-- wherein R₃ is H, alkyl, or cycloalkyl-loweralkyl;and X is a halogen; and the acid addition salts thereof.

The term "alkyl" as used herein refers to both straight and branchedchain alkyl radicals having 1 to 8 carbon atoms and which includemethyl, ethyl, n-butyl, n-pentyl, iso-pentyl, hexyl and the like.

As used herein, the term "loweralkyl-alkyl" means saturated monovalentaliphatic radicals including straight and branched chain radicals offrom 1 to 6 carbon atoms, as illustrated by, but not limited to methyl,ethyl, propyl, iso-propyl, butyl, sec-butyl, amyl, hexyl, and the like.

As used herein, the term "cycloalkyl" means cyclic, saturated, aliphaticradicals of from 3 to 8 carbon atoms, as illustrated by, but not limitedto, cyclopropyl, cyclobutyl, 2-methyl-cyclobutyl, cyclohexyl,4-methylcyclohexyl, cyclooctyl and the like.

The term "halogen" includes chlorine, fluorine, bromine and iodine.

The compounds of this invention exhibit antibacterial activity and arequite useful as a means of removing disease causing bacteria from livingorganisms.

The compounds of this invention are also useful in potentiating theantibacterial effect of sulfonamides such as sulfadiazine,sulfamethoxazole, sulfamethazine, and sulfamerazine. The combination ofthese compounds with sulfonamides may increase the antibacterialactivity of the sulfa drug by as much as 30-fold. The ratio ofcompound:sulfonamide in these mixtures may vary over a wide range. Forexample, the ratio may vary from 1:1 to 1:20. These mixtures may beformulated in dosage forms convenient for administration to patients.

The present compounds may be made according to the following generalprocess scheme: ##STR3## wherein R₁ is H or loweralkyl; R₂ is H,loweralkyl or R₃ CO-- wherein R₃ is H, alkyl, cycloalkyl, loweralkyl;and X is a halogen.

The above process is carried out by first treating an aldehyde ofFormula II with 3-anilinopropionitrile in the presence of an alkalimetal lower alkoxide. The alkali metal alkoxide is preferably sodiummethoxide or sodium ethoxide. The reaction temperature is determined bythe temperature of the refluxing solvent which is preferably methanol orethanol. The resulting substituted acrylonitrile is usually not isolatedbut instead is treated in situ with guanidine to give the pyrimidine ofFormula I. Alternatively, the substituted acrylonitrile can be treatedwith anhydrous ethanolic hydrochloride to give an imidate ester whichthen affords the pyrimidine of Formula I after treatment with guanidine.This modified procedure affords somewhat better yields and purerproducts than the unmodified procedure.

The aldehydes of formula II are prepared according to the scheme below:##STR4## wherein R₁, R₂, and X are as defined above.

The intermediates such as the benzoic acid methyl ester represented byformula III may be prepared by the method described in U.S. Pat. No.3,801,636. These intermediates are useful in the preparation of thedesired pyrimidine compounds.

Some of the compounds produced by the general process scheme,illustrated above, which come within the scope of Formula (I) are:

2,4-Diamino-5-(4-amino-3,5-dichlorobenzyl)pyrimidine of the formula##STR5##

2,4-Diamino-5-(3,5-dichloro-4-methylaminobenzyl)pyrimidine of theformula ##STR6##

2,4-Diamino-5-(3,5-dichloro-4-ethylaminobenzyl)pyrimidine of the formula##STR7##

2,4-Diamino-5-(3,5-dichloro-4-dimethylaminobenzyl)pyrimidine of theformula ##STR8##

2,4-Diamino-5-(4-acetamido-3,5-dichlorobenzyl)pyrimidine of the formula##STR9##

The following examples will serve to further illustrate the preparationof the present compounds and the advantages of the present invention.

EXAMPLE I 2,4-Diamino-5-(4-Amino-3,5-Dichlorobenzyl)Pyrimidine ##STR10##

A solution of sodium ethoxide in ethanol was prepared by dissolving 0.58g. (0.025 mole) of sodium in 250 ml. of absolute ethanol. To thissolution was added 15.4 g. (0.105 mole) of 3-anilinopropionitrilefollowed by 16.2 g. (0.085 mole) of 4-amino-3,5-dichlorobenzaldehyde.This mixture was heated under reflux for 8 hours. The imidate ester wasformed by first cooling the above mixture to 10° and then adding 0.7 ml.of 0.4 N ethanolic-HCl. Formation was complete after stirring for 1 hourat 10° C. Guanidine free base, prepared by adding 22.9 g. (0.24 mole) ofguanidine hydrochloride to 5.52 g. (0.24 mole) of sodium in 90 ml. ofabsolute ethanol, was added to the mixture containing the imidate ester.After stirring and heating under reflux for 5 hours, the sodium chloridewas removed by filtration and the filtrate concentrated in vacuo untilits volume was diminished by one-half. Cooling induced the product toprecipitate. Crystallization from isopropanol afforded 3.6 g. ofmaterial having a melting point of 241° C.

Analysis Calcd. for C₁₁ H₁₁ Cl₂ N₅ : C, 46.49; H, 3.90; N, 24.65 Found:C, 42.41; H, 3.42; N, 24.35

In Table 1, the physical data of other compounds produced by the methoddescribed above, are shown. The compounds, as identified, are thoserepresented respectively by formulas VI and VIII:

                                      TABLE 1                                     __________________________________________________________________________                          Theory   Found                                          Compound                                                                            M.P.    Formula C  H  N  C  H  N                                        __________________________________________________________________________    VI    202-203° C.                                                                    C.sub.12 H.sub.13 Cl.sub.2 N.sub.5                                                    48.5                                                                             3.3                                                                              23.5                                                                             48.7                                                                             4.3                                                                              23.5                                     VIII  193-194° C.                                                                    C.sub.13 H.sub.15 Cl.sub.2 N.sub.5                                                    50.0                                                                             4.8                                                                              22.4                                                                             50.1                                                                             4.7                                                                              22.3                                     These compounds are useful as antibacterial agents.                           __________________________________________________________________________

EXAMPLE II 2,4-Diamino-5-(3,5-Dichloro-4-Ethylaminobenzyl)Pyrimidine##STR11##

A solution of sodium ethoxide in ethanol was prepared by dissolving 1.15g. (0.05 mole) of sodium in 500 ml. of absolute ethanol. To thissolution was added 30.7 g. (0.209 mole) of 3-anilinopropionitrilefollowed by 36.4 g. (0.167 mole) of3,5-dichloro-4-ethylaminobenzaldehyde. This mixture was heated underreflux for 8 hours. To this was added guanidine free base, prepared byadding 45.8 g. (0.48 mole) of guanidine hydrochloride to 11.04 g. (0.48mole) of sodium in 100 ml. of absolute ethanol. The total mixture wasstirred and heated under reflux for 5 hours and then cooled to roomtemperature. The sodium chloride was removed by filtration and thefiltrate concentrated in vacuo until its volume was diminished byone-half. Cooling induced the product to precipitate. Crystallizationfrom isopropanol offered 4.8 g. of material having a melting point of201°-202° C.

Analysis Calcd. for: C₁₃ H₁₅ Cl₂ N₅ : C, 50.01; H, 4.84; N, 22.43 Found:C, 49.73; H, 4.87; N, 22.50

EXAMPLE II 2,4-Diamino-5-(4-Acetamido-3,5-Dichlorobenzyl)Pyrimidine##STR12##

A solution of sodium ethoxide in ethanol was prepared by dissolving 1.15g. (0.05 mole) of sodium in 500 ml. of absolute ethanol. To this mixturewas added 30.7 g. (0.209 mole) of 3-anilinopropionitrile followed by38.8 g. (0.167 mole) of 4-acetamido-3,5-dichloro-benzaldehyde. Thismixture was heated under reflux for 8 hours. Guanidine free base,prepared by adding 45.8 g. (0.48 mole) of guanidine hydrochloride to11.04 g. (0.48 mole) of sodium in 100 ml. of absolute ethanol, was addedto the mixture above. The combined reaction mixture was stirred andheated under reflux for 5 hours and then cooled to room temperature. Thesodium chloride was removed by filtration and the filtrate concentratedin vacuo until its volume was diminished by one-half. Cooling induced anoil to form. The oil was triturated with CH₂ Cl₂ and the CH₂ Cl₂ wasthen evaporated in vacuo leaving a solid. Crystallization of the solidfrom ethanol afforded 3.1 g. of material having a melting point of251°-253° C.

Analysis Calcd. for C₁₃ H₁₃ Cl₂ N₅ O: C, 47.87; H, 4.02; N, 21.47 Found:C, 48.05; H, 3.89; N, 21.37

EXAMPLE IV

Several tests have been made to determine the activity of the presentpyrimidine compounds as antibacterial agents. The tests have includedthe following compounds:

(VII) 2,4-Diamino-5-(3,5-dichloro-4-ethylaminobenzyl)pyrimidine;

(VI) 2,4-Diamino-5-(3,5-dichloro-4-methylaminobenzyl)pyrimidine;

(VIII) 2,4-Diamino-5-(3,5-dichloro-4-dimethylaminobenzyl)pyrimidine;

(V) 2,4-Diamino-5-(4-amino-3,5-dichlorobenzyl)pyrimidine.

Test 1

The four compounds, i.e., compounds V, VI, VII, VIII, were testedagainst a series of strains including:

Escherichia coli Juhl and Escherichia coli 3100. The method used was atwo-fold tube dilution test in 5 ml. amounts of minimal medium. Theinoculum was 0.1 ml. of a 1:1T dilution of a 24 hour broth culture. Thetest was incubated at 37° C. for 18 hours. The results of the test areprovided in Table 2 below.

                  Table 2                                                         ______________________________________                                        In Vitro Antimicrobial Activity                                                               Minimum Inhibitory                                                            Concentration (Mcg./Ml.)                                                      Compounds                                                     Organism          VII     VI      VIII  V                                     ______________________________________                                        Escherichia coli Juhl                                                                            3.1    6.2     6.2   0.78                                  Escherichia coli 3100                                                                            6.2    3.1     6.2   0.39                                  Enterobacter aerogenes 13048                                                                    25      3.1     3.1   1.56                                  Proteus mirabilis Finland #9                                                                    50      12.5    25    25                                    Salmonella typhimurium Ed  9                                                                    12.5    3.1     6.2   3.1                                   Klebsiella pneumoniae 8045                                                                      12.5    6.2     6.2   1.56                                  ______________________________________                                    

Test 2

A similar test was made on other strains with the compounds (V, VI, VIIand VIII) in 5 ml. amounts of Mueller Hinton Medium. The results of thistest are provided in Table 3 below:

                  Table 3                                                         ______________________________________                                                        Minimum Inhibitory                                                            Concentration (Mcg./Ml.)                                                      (Mueller Hinton Medium)                                                       Compounds                                                     Organism          VII     VI      VIII  V                                     ______________________________________                                        Staphylococcus aureus 209P                                                                      3.1     3.1     1.56  6.2                                   Staphylococcus aureus Smith                                                                     6.2     6.2     3.1   25                                    Streptococcus pyogenes C203                                                                     12.5    6.2     3.1   6.2                                   ______________________________________                                    

Test 3

Compounds V - VIII were tested and compared with trimethoprim, ascontrol, against two external strains, (Escherichia coli) a sensitiveNo. 114 and a No. 114 carrying a resistant (R) factor which mediates thesynthesis of an altered dihydrofolate reductase; S. G. R. Amyes and J.T. Smith, Biochem. and Biophys., Res. Comm., 58, 412 (1974).

The method used was a two-fold tube dilution test in 5 ml. amounts ofminimal medium. The inoculum was 0.1 ml. of a 1:1T dilution of a 24 hourbroth culture. The test was incubated at 37° C. for 18 hours.

The results of the test are shown below in Table 4.

                  Table 4                                                         ______________________________________                                        In Vitro Antimicrobial Activity vs. Trimethoprim                              Resistant E. Coli                                                                       Minimum Inhibitory Concentration                                              (Mcg./Ml.)                                                                      E. Coli 114   E. Coli                                             Compound    Sensitive     (Resistant)                                         ______________________________________                                        Trimethoprim                                                                              0.2           3000                                                VII         3.1           200                                                 VI          1.56          >400                                                VIII        6.2           100                                                 V           1.56          400                                                 ______________________________________                                    

As shown above, the presence of the resistant (R) factor in E. Coli 114caused an increase in trimethoprim resistance of several thousand-fold,whereas the increase in resistance to compounds V - VIII issignificantly less than this.

Test 4

The potentiation properties of the compounds, i.e., compounds V - VIIIin various combinations with sulfa compounds, i.e., sulfonamides(sulfamethoxazole, sulfadiazine, sulfamethazine and sulfamerazine) weretested.

The method was a two-fold tube dilution test using BBL Nutrient broth in5 ml. amounts. The inoculum was 0.1 ml. of a 10⁻³ dilution 24 hour brothculture of Escherichia Coli 3100. The test was incubated at 37° C. for18 hours.

The results of the test are provided below in Table 5.

                  Table 5                                                         ______________________________________                                        In Vitro Potentiation of Sulfonamides vs. E. Coli 3100                        ______________________________________                                                             Compound/                                                Compound                                                                              Sulfonamide  Sulfa Ratio Potentiation*                                ______________________________________                                        VII     Sulfamethoxazole                                                                           1:5         3.1 X                                        VII     Sulfamethoxazole                                                                           1:1         4.0 X                                        VII     Sulfadiazine 1:5         6.2 X                                        VII     Sulfadiazine 1:1         8.0 X                                        VII     Sulfamethazine                                                                             1:5         3.2 X                                        VII     Sulfamethazine                                                                             1:1         4.0 X                                        VII     Sulfamerazine                                                                              1:5         3.2 X                                        VII     Sulfamerazine                                                                              1:1         8.0 X                                        VI      Sulfadiazine 1:5         3.1 X                                        VI      Sulfadiazine 1:1         15.9 X                                       VIII    Sulfadiazine 1:5         1.6 X                                        VIII    Sulfadiazine 1:1         8.0 X                                        V       Sulfadiazine 1:5         3.1 X                                        V       Sulfadiazine 1:1         31.0 X                                       ______________________________________                                         *The potentiation effect shown is determined by measuring the minimum         inhibitory concentration (MIC) of the sulfonamide alone and in combinatio     with the compound at the indicated ratios.                               

Test 5 Acute Mouse Protection Test

The test bacteria were transferred to Brain Heart Infusion Broth andincubated at 37° C. for 18 hours. Log dilutions of the organism weremade in BHI broth and mixed with 3% hog gastric mucin.

Female, Swiss albino mice, 18 - 20 grams, were injectedintraperitoneally with 0.75 ml. of inoculum.

The optimum dilution was one in which each mouse received 10 - 10 LD₅₀'s of the infecting organism.

The test compound was diluted in such a way that there were 5 druglevels (each level is half as concentrated as the one above) in eachtest. Ten infected mice were treated with each level of drug. The micewere medicated 1 and 5 hours post-infection.

The mouse mortality was observed for 7 days.

An approximate CD₅₀ in mg/kg of the in vivo activity of combinations ofa sulfa drug and compounds V - VIII is recorded below in Table 6.

                  Table 6                                                         ______________________________________                                        In Vivo Activity of Combinations In Acute Mouse Protection                    ______________________________________                                        Test                                                                                    CD.sub.50 (Mg./Kg.)                                                 ______________________________________                                                    Sulfa    Sulfa    Sulfa  Sulfa                                                Cmpd.    Cmpd.    Cmpd.  Cmpd.                                    Organism    VII      VI       VIII   V                                        ______________________________________                                        E. Coli (Juhl)                                                                            175/35   150/30   225/45 225/45                                   E. Coli (48)                                                                              10/2     15/3     15/3   --                                       P. Mirabilis                                                                              150/30   300/60   400/80 150/30                                   P. Vulgaris 10/2     10/2     10/2    5                                       ______________________________________                                         Sulfa = Sulfamethoxazole                                                      The data shows that the combinations are effective in curing lethal           infections in mice.                                                      

We claim:
 1. An antibacterial composition containing as theantibacterial components a compound of the formula ##STR13## wherein R₁is H or loweralkyl; R₂ is H, loweralkyl or R₃ CO wherein R₃ is H orloweralkyl, and x is a halogen or a pharmaceutically-acceptable acidaddition salt thereof; and a sulfonamide selected from the groupconsisting of sulfadiazine, sulfamethoxazole, sulfamethazine andsulfamerazine, said components being present in a ratio of between 1:1and 1:20.
 2. A composition according to claim 1, wherein said compoundis 2,4-diamino-5-(3,5-dichloro-4-ethylaminobenzyl)pyrimidine and saidsulfonamide is sulfamethoxazole.
 3. A composition according to claim 1,wherein said compound is2,4-diamino-5-(4-amino-3,5-dichlorobenzyl)pyrimidine and saidsulfonamide is sulfadiazine.
 4. A composition according to claim 1,wherein said compound is2,4-diamino-5-(3,5-dichloro-4-methylaminobenzyl)pyrimidine and saidsulfonamide is dulfadiazine.
 5. A composition according to claim 1,wherein said compound is2,4-diamino-5-(3,5-dichloro-4-dimethylaminobenzyl)pyrimidine and saidsulfonamide is sulfadiazine.
 6. A composition according to claim 1,wherein said compound is2,4-diamino-5-(3,5-dichloro-4-ethylaminobenzyl)pyrimidine and saidsulfonamide is sulfamerazine.
 7. A composition according to claim 1,wherein said compound is2,4-diamino-5-(3,5-dichloro-4-ethylaminobenzyl)pyrimidine and saidsulfonamide is sulfamethazine.
 8. A composition according to claim 1,wherein said compound is2,4-diamino-5-(3,5-dichloro-4-ethylaminobenzyl)pyrimidine and saidsulfonamide is sulfadiazine.